1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using a Zea mays glycine rich protein promoter.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), and sucrose synthase (Yang and Russell, 1990).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991), (xcex1-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), and chalcone synthase promoters (Franken et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
A class of genes which are expressed in an inducible manner are glycine-rich proteins (GRPs). GRPs are a class of proteins characterized by their high content of glycine residues, which often occur in repetitive blocks (Goddemeier et al., 1998). Many GRPs are thought to be structural wall proteins or RNA-binding proteins (Mar Alba et al., 1994). Genes encoding glycine rich proteins have been described, for example, from maize (Didierjean et al., 1992; Baysdorfer, Genbank Accession No. AF034945) sorghum (Cretin and Puigdomenech, 1990), and rice (Genbank Accession No. AF009411). One maize GRP coding sequence in particular, the expression of which was found to be water and wounding stress-inducible, was isolated by Gomez et al., (1988), the sequence of which is given by Genbank Accession No. GI:22312. However, this reference failed to provide the sequence of the promoter of the gene.
Although the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter sequences with beneficial expression characteristics. The number of effective promoters available for use with transgenes in maize is not abundant. New promoters, especially promoters that will express differentially in maize tissues, are spatially and/or temporally expressed, or are induced to express by different environmental signals, would be useful. Such expression specific promoters could be useful in minimizing yield drag and other potential adverse physiological effects on maize growth and development that might be encountered by high-level, non-inducible, constitutive expression of a transgenic protein in a plant. A wider range of effective promoters also would make it possible to introduce multiple transgenes into a plant, each fused to a different promoter, thereby minimizing the risk of DNA sequence homology dependent transgene inactivation (co-suppression). Therefore, there is a great need in the art for the identification of novel inducible promoters which can be used for the high-level expression of selected transgenes in economically important crop plants.
In one aspect, the invention provides an isolated nucleic acid comprising a maize GRP promoter. Still further provided by the invention is a maize GRP promoter isolatable from the nucleic acid sequence of SEQ ID NO:1. In particular embodiments, a GRP promoter in accordance with the invention may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 400 to about 3536, about 750 to about 3536, about 1000 to about 3536, about 1500 to about 3536, about 2000 to about 3536, about 2500 to about 3536, or about 3000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1, as well as all lengths of contiguous nucleotides within such sizes. In further embodiments, the GRP promoter may comprise the nucleic acid sequence of SEQ ID NO:1.
An isolated nucleic acid comprising a maize GRP promoter in accordance with the invention may further comprising an enhancer, for example, an intron. In one embodiment, the intron is a rice actin 1 intron or rice actin 2 intron. The isolated nucleic acid may further comprise a terminator, such as a rbcS terminator.
In another aspect, the invention provides a transgenic plant stably transformed with a selected DNA comprising a maize GRP promoter. In particular embodiments of the invention, the maize GRP promoter may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 400 to about 3536, about 750 to about 536, about 1000 to about 3536, about 1500 to about 3536, about 2000 to about 3536, about 2500 to about 3536, or about 3000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. In one embodiment of the invention, the GRP promoter comprises the nucleic acid sequence of SEQ ID NO:1.
The selected DNA may further comprise any additional desired sequences. In one embodiment of the invention, the selected DNA further comprises a selected heterologous coding region operably linked to the maize GRP promoter. Potentially any coding sequence could be employed with the maize GRP promoter, including a selected coding region which encodes a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. Where the selected coding region encodes a protein imparting a selectable marker phenotype, the protein may be selected from, for example, the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase. The selected coding region may be operably linked to a terminator, for example, an rbcS terminator, including a rice rbcS terminator. Benefit may also be realized by including an enhancer with the selected DNA. Examples of such an enhancer include the rice actin 1 intron and rice actin 2 intron.
The selected DNA may further comprise DNA from a cloning vector, such as plasmid DNA, or alternatively, may have been introduced as an expression cassette isolated from such vector DNA. The selected DNA may also comprise a sequence encoding a signal peptide. Examples of signal peptides that could be sued include a peroxisomal targeting peptide or a chloroplast transit peptide. Examples of a chloroplast transit peptide include the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide.
A transgenic plant comprising a selected DNA in accordance with the invention may be potentially any type of plant, including a monocotyledonous or dicotyledonous plant. Examples of monocotyledonous plants include wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane. In one embodiment of the invention, the monocotyledonous plant is maize. Examples of dicotyledonous plants include tobacco, tomato, potato, soybean, cotton, canola, alfalfa, sunflower, and cotton. In one embodiment of the invention the dicotyledonous plant is a soybean plant. The transgenic plant prepared in accordance with the invention may be of any generation, including a fertile R0 transgenic plant as well as seeds thereof, wherein the seed comprises the selected DNA. Also included within the invention are progeny plants of any generation such a fertile R0 transgenic plant, wherein the progeny plant comprises said selected DNA, as well as seed of a progeny plant, wherein said seed comprises said selected DNA.
In yet another aspect, the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a maize GRP promoter, wherein the maize GRP promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1; (ii) crossing the fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. The invention also includes a seed or seeds of such a crossed fertile transgenic plant, wherein said seed comprises said selected DNA. The crossed fertile transgenic plant may be potentially any type of plant, including a monocotyledonous or dicotyledonous plant. Examples of monocotyledonous plants include wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane. In one embodiment of the invention, the monocotyledonous plant is maize. Examples of dicotyledonous plants include tobacco, tomato, potato, soybean, cotton, canola, alfalfa, sunflower, and cotton. In one embodiment of the invention the dicotyledonous plant is a soybean plant. The selected DNA may have been inherited through a parent used a male or a female at any given generation. In one embodiment of the invention, the second plant is an inbred plant. Where the second plant is an inbred, the crossed fertile transgenic plant may be a hybrid, or also inbred where it is crossed with itself.
The crossed fertile transgenic plant may comprise any of the maize GRP promoter compositions provided by the invention. In one embodiment of the invention, the maize GRP promoter comprises about 95 to 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 400 to about 3536, about 750 to about 3536, about 1000 to about 3536, about 1500 to about 3536, or about 2000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. In another embodiment of the invention, the fertile transgenic plant may comprise the full length of the nucleic acid sequence of SEQ ID NO:1, or a derivative thereof. The maize GRP promoter may further be operably linked to a selected heterologous coding region. Potentially any coding region could be used, including a selected coding region which encodes a protein selected from the group consisting of a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. The plant may further comprise a selected DNA comprising an enhancer, for example, a rice actin 1 intron and rice actin 2 intron. The selected coding region may be operably linked to a terminator, for example, an rbcS terminator such as a rice rbcS terminator.
In still yet another aspect, the invention provides a method of expressing a selected protein in a transgenic plant comprising the steps of: (i) obtaining a construct comprising a selected coding region operably linked to a maize GRP promoter, wherein the maize GRP promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1; (ii) transforming a recipient plant cell with the construct; and (iii) regenerating a transgenic plant expressing the selected protein from the recipient plant cell. In one embodiment of the invention, the transgenic plant is fertile. The method may further comprise the step of obtaining seed from the fertile transgenic plant, and may still further comprise obtaining a progeny plant of any generation from the fertile transgenic plant. The transforming may be achieved in any manner, including a method selected from the group consisting of microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation. In one embodiment of the invention, the step of transforming comprises microprojectile bombardment. The recipient plant cell may be of any type desired, including from a monocotyledonous or dicotyledonous plant. Examples of such a monocotyledonous plant include wheat, maize, rye, rice, turfgrass, oat, barley, sorghum, millet, and sugarcane. In one embodiment of the invention, the monocotyledonous plant is a maize plant. Examples of dicotyledonous plants include tobacco, tomato, potato, soybean, canola, sunflower, alfalfa and cotton.
Any type of selected coding region may be used with the maize GRP promoter, including a coding region encoding a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. The construct used may further comprise any additional sequences desired, including an enhancer. Exemplary enhancers include the rice actin 1 intron and rice actin 2 intron. The selected coding region may be operably linked to a terminator, for example, an rbcS terminator including a rice rbcS terminator.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: (i) obtaining a transgenic plant comprising a selected DNA comprising a maize GRP promoter, wherein the maize GRP promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1; and (ii) crossing the transgenic plant with itself or a second plant. The transgenic plant may be of potentially any species, including monocotyledonous or dicotyledonous plants. Examples of such monocotyledonous plants include wheat, maize, oat, barley, rye, rice, turfgrass, sorghum, millet and sugarcane. In one embodiment of the invention, the monocotyledonous plant is a maize plant. Examples of dicotyledonous plants include tobacco, tomato, potato, soybean, canola, sunflower, alfalfa and cotton. The selected DNA may comprise any of the maize GRP promoter compositions provided by the invention, and may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 5400 to about 3536, about 600 to about 3536, about 800 to about 3536, about 1000 to about 3536, about 1500 to about 3536, or about 2000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The maize GRP promoter may also comprise the nucleic acid sequence of SEQ ID NO:1, or a derivative thereof, such as a deletion mutant.
In one embodiment of the invention, the transgenic plant is crossed with the second plant. The second plant may be an inbred plant. The second plant may be an inbred plant. The method may further comprise the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. The progeny plant can inherit the selected DNA through a parent used as a male or female at any given generation. In one embodiment of the invention, the second plant and the third plant are of the same genotype, and further, may be inbred plants. The selected DNA may comprise potentially any coding region, for example, a coding region which encodes a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. The selected DNA may further comprise a genetic element which enhances the expression of the protein in the transgenic plant, including a rice actin 1 intron and the rice actin 2 intron.
In still yet another aspect, the invention provides a transgenic plant cell stably transformed with a selected DNA comprising a maize GRP promoter, wherein said maize GRP promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1. The plant cell may comprise any of the maize GRP promoter compositions provided by the invention, and may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 200 to about 3536, about 400 to about 3536, about 750 to about 3536, about 1000 to about 3536, about 1500 to about 3536, or about 2000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The maize GRP promoter may also comprise the nucleic acid sequence of SEQ ID NO:1, or a derivative thereof. The selected DNA may further comprise potentially any selected coding region operably linked to the maize GRP promoter, including a selected coding region which encodes a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. Where the selected coding sequence encodes a protein which imparts a selectable marker phenotype, exemplary coding sequences encode a protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase. The selected coding region may be operably linked to a terminator, such as an rbcS terminator, including a rice rbcS terminator. The selected DNA may also comprise an enhancer, including a rice actin 1 intron and rice actin 2 intron.
The selected DNA may also comprise vector DNA, such as plasmid DNA, or may be isolated from such DNA. The selected DNA also may comprise a sequence encoding a signal peptide, including a peroxisomal targeting peptide or a chloroplast transit peptide. Exemplary transit peptides include a transit peptide selected from the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide. The transgenic plant cell may be of any species, and may be from a monocotyledonous or dicotyledonous plant. Exemplary monocotyledonous plants include wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane. In one embodiment of the invention, the plant is a maize plant. Exemplary dicotyledonous plants include tobacco, tomato, potato, soybean, cotton, canola, alfalfa and sunflower. In one embodiment of the invention, the dicotyledonous plant is a soybean plant.